P-type ATPases, which include the highly homologous NalK pump and sarco/endoplasmic reticulum caldum pump (SERCA), are the enzymes responsible for maintaining electrolyte homeostasis critical for cell function. These pumps use the energy from ATP hydrolysis to power the uphill transmembrane movement of ions. Their defining feature is the transfer of a phosphate from ATP to the pump to form a high-energy phosphointermediate. The recent high-resolution structure of SERCA in two conformations provides important new information for understanding the structure-function relationship of P-type ATPases. However, neither structure contains the unique phosphointermediate form of this class of ion pumps. This grant focuses on the Na,K pump because it offers an important advantage for elucidating the molecular coupling between ion movements and ATP hydrolysis. The Na pump advantage is that K is the counter cation vs. H for SERCA. Both crystallographically and biochemically, following the K ion is much easier than following the transported H. "That is, almost all K effects occur at the cation transport site, whereas proton effects can be the result of several amino acid titrations in addition to binding at the transport sites. It is impossible to study SERCA in the absence of protons, whereas one can easily study the Na pump in the absence of K. P-type pumps have three major cytoplasmic domains: the N domain which binds nucleotides, the P domain which contains the aspartic acid residue that accepts the forms the catalytic phosphointermediate (EP), and the A domain which is important for both catalytic phosphorylation and dephosphorylation. When the Na pump is catalytically phosphorylated on Asp369, the N and P domains must necessarily be in close contact to allow Asp369 to attack the terminal phosphate on ATP. Where are the N and P domains during other enzyme conformations? Our aim is to track the relative motion of these domains throughout the transport cycle using biochemical and fluorescence-based assays. Our aims are to determine the movements of the N domain during the pump cycle, determine the status of the phosphointermediate during the pump cycle, again focusing on the extracellular binding and release steps, and to examine the influence of N domain ligands on the status of the transmembrane domains.